Amelogenin Interacts with Cytokeratin-5 in Ameloblasts during
Received for publication, October 31, 2002, and in revised form, March 24, 2003
Published, JBC Papers in Press, March 25, 2003, DOI 10.1074/jbc.M211184200
Rajeswari M. H. Ravindranath‡, Rajam M. Basilrose, Sr., Naren H. Ravindranath,
and Bhavapriya Vaitheesvaran
From the Center for Craniofacial Molecular Biology, School of Dentistry, University of Southern California,
Los Angeles, California 90033-1004
The enamel protein amelogenin binds to GlcNAc
(Ravindranath, R. M. H., Moradian-Oldak, R., and
Fincham, A.G. (1999) J. Biol. Chem. 274, 2464–2471) and
to the GlcNAc-mimicking peptide (GMp) (Ravindranath,
R. M. H., Tam, W., Nguyen, P., and Fincham, A. G. (2000)
J. Biol. Chem. 275, 39654–39661). The GMp motif in the
N-terminal region of the cytokeratin 14 of ameloblasts
binds to trityrosyl motif peptide (ATMP) of amelogenin
(Ravindranath, R. M. H., Tam, W., Bringas, P., Santos, V.,
and Fincham, A. G. (2001) J. Biol. Chem. 276, 36586–
36597). K14 (Type I) pairs with K5 (Type II) in basal
epithelial cells; GlcNAc-acylated K5 is identified in
ameloblasts. Dosimetric analysis showed the binding
affinity of amelogenin to K5 and to GlcNAc-acylated-
positive control, ovalbumin. The specific binding of
[3H]ATMP with K5 or ovalbumin was confirmed by Scat-
chard analysis. [3H]ATMP failed to bind to K5 after re-
moval of GlcNAc. Blocking K5 with ATMP abrogates the
K5-amelogenin interaction. K5 failed to bind to ATMP
when the third proline was substituted with threonine,
as in some cases of human X-linked amelogenesis imper-
fecta or when tyrosyl residues were substituted with
phenylalanine. Confocal laser scan microscopic obser-
vations on ameloblasts during postnatal (PN) growth of
the teeth showed that the K5-amelogenin complex mi-
grated from the cytoplasm to the periphery (on PN day
1) and accumulated at the apical region on day 3. Secre-
tion of amelogenin commences from day 1. K5, similar to
K14, may play a role of chaperone during secretion of
amelogenin. Upon secretion of amelogenin, K5 pairs
with K14. Pairing of K5 and K14 commences on day 3
and ends on day 9. The pairing of K5 and K14 marks the
end of secretion of amelogenin.
Ameloblasts synthesize and secrete the proteins involved in
the microstructure and biomineralization of the enamel.
Amelogenin constitutes 90% of the total enamel proteins se-
creted by ameloblasts. It is a non-glycosylated single polypep-
tide of about 180 amino acids (1–3). The N-terminal 45 amino
acid residues are referred to as tyrosine-rich amelogenin
polypeptide (TRAP).1The C-terminal 13-amino acid sequence
in the TRAP region called “amelogenin tyrosyl motif peptide”
(ATMP: PYPSYGYEPMGGW) possesses unique ligand-binding
properties, in that it binds specifically to N-acetylglucosamine
(GlcNAc) (4) and GlcNAc-mimicking peptides (GMps) (5). Mu-
tations in the ATMP sequence are observed in the human
X-linked amelogenesis imperfecta (AI) (6). The mutated forms
of ATMP failed to bind with GlcNAc or the GMp (4, 5). Loss-
of-function “mutations” of ATMP correlated well with the loss
of amelogenin-ligand interaction.
A GMp motif is found in the N-terminal region of Type I
cytokeratin 14 (K14), a differentiation marker for ameloblasts
prior to amelogenin synthesis (7). The purified or recombinant
amelogenin and TRAP bind to K14 in vitro dosimetrically (8).
Scatchard analysis confirms the specific interaction between
K14 and ATMP in vitro. GlcNAc and GMp blocked binding of
rM179 or ATMP with K14. Mutated ATMP failed to bind to
K14 when the third proline was substituted with threonine, as
in some cases of human X-linked AI (6) or when tyrosyl resi-
dues were substituted with phenylalanine. Amelogenin co-as-
sembled with K14 in the perinuclear region of ameloblasts on
day 0. The K14-amelogenin complex migrated to the apical
region of the ameloblasts on day 1 and accumulated there
between days 3 and 5 and collapsed on day 9. Autoradiography
with [3H]ATMP and [3H]GMp corroborated the dissociation of
amelogenin and K14 at the Tomes’ process of the ameloblast,
suggesting that K14 plays a chaperone role for nascent
amelogenin polypeptide during secretion of amelogenin (8).
K14 (Type I) pairs with Type II K5 in basal epithelial cells.
Such pairing of Type I and Type II cytokeratins are known to
occur during epithelial cell differentiation (9). K5 is also pres-
ent in ameloblasts (10, 11). The pairing of K5 with K14 requires
N-terminal regions of both cytokeratins (12). We hypothesize
that, if the N-terminal region (GMp motif) of K14 is bound to
the ATMP motif of amelogenin, it may not pair with K5 until
amelogenin is disassociated from it. Furthermore, we hypoth-
esize that K5, per se, might also bind to amelogenin, because it
may carry GlcNAc at the N-terminal residues similar to other
Type II keratins (K8) (13). If K5 were to bind to amelogenin, it
might define the relative role of Type I and II cytokeratins in
secretion of amelogenin. If pairing of K14 and K5 were to occur
in ameloblasts, it would be important to define the event in the
context of amelogenin secretion and enamel formation.
We present evidence to show that K5 binds to amelogenin,
leading to the secretion of amelogenin and pairing with K14.
The in vitro and in situ observations, during different stages of
* This work was supported by NIDR, National Institutes of Health
Grant DE-03660 (to R. M. H. R.). The costs of publication of this article
were defrayed in part by the payment of page charges. This article must
therefore be hereby marked “advertisement” in accordance with 18
U.S.C. Section 1734 solely to indicate this fact.
‡ To whom correspondence should be addressed. Tel.: 323-442-3171;
Fax: 323-442-2981; E-mail: email@example.com.
1The abbreviations used are: TRAP, tyrosine-rich amelogenin
polypeptide; ATMP, ameloblasts binds to trityrosyl motif peptide; Glc-
NAc, N-acetylglucosamine; GMp, GlcNAc-mimicking peptide; AI,
amelogenesis imperfecta; PN, postnatal; BME, buffered urea-?-
mercaptoethanol; PVDF, polyvinylidene difluoride; PBS, phosphate-
buffered saline; HSA, human serum albumin; BSA, bovine serum albu-
min; FITC, fluorescein isothiocyanate; TRITC, tetramethylrhodamine
isothiocyanate; GD2, disialoganglioside 2; GD3, disialoganglioside 3;
GM2, monosialoganglioside 2; GD1b, disialoganglioside 1b.
THE JOURNAL OF BIOLOGICAL CHEMISTRY
© 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 278, No. 22, Issue of May 30, pp. 20293–20302, 2003
Printed in U.S.A.
This paper is available on line at http://www.jbc.org
by guest on December 22, 2015
enamel formation, define the interactions among amelogenin
and cytokeratins. The pairing of K14 with K5 does occur after
secretion of amelogenin and just prior to the disintegration of
cytokeratins. These findings are novel and critical, and they
define the relative role of cytokeratins in the secretion of
amelogenin during enamel formation. The results are impor-
tant for understanding the events taking place during secretion
of amelogenin and enamel formation, and the development of
corrective measures to prevent abnormal enamel development
in genetic disorders like AI and different kinds of epidermolysis
bullosa caused by mutations in K14 or K5 (14–18).
Mice—All observations were made on Swiss Webster mice at differ-
ent developmental stages ranging from newborn day “0” through post-
natal days (PN) 1, 3, 5, 7, and 9. A total of 25 normal, healthy, female
pregnant Swiss Webster mice (Charles River Breeding) were used to
obtain a sufficient number of litters as was carried out previously (8).
The Institutional Animal Care and Use Committee (Los Angeles, CA)
approved all protocols involving mice.
Isolation and Purification of K5—We have isolated and purified K5
from HeLa cells (19). We have obtained HeLa cells (CCL-2 obtained
from ATCC) and have grown them in Eagle’s minimum essential me-
dium containing 10% fetal bovine serum at 37 °C. The cells were col-
lected and frozen at ?20 °C. The proteins were extracted as previously
described (20, 21). The keratin fraction was obtained from cells that
were homogenized in equal volumes of ice-cold Tris-Triton buffer (25
mM Tris-HCl, pH 7.4, 0.5% Triton X-100, 1 mM EDTA, 1 mM of phen-
ylmethylsulfonyl fluoride). The extract was centrifuged at 10,000 ? g
for 2 min. The pellet was extracted three times with Tris-Triton buffer.
The supernatant fractions contain most of the soluble cellular proteins,
including many membrane proteins. The pellet was washed in Tris
buffer and then dispersed in double the volume of Tris-HCl, pH 7.4,
with 1 M KCl, incubated for 30 min at 37 °C, and once again centrifuged
for 10,000 ? g for 30 min. The final pellet was solubilized in about 1.5
volumes of buffered urea-?-mercaptoethanol (BME) solution (25 mM
Tris-HCl, pH 7.4, 9 M urea, 1 mM EDTA, 100 mM BME, 1 mM phenyl-
methylsulfonyl fluoride). The extracted proteins were purified by the
USC Microchemical Core Laboratory, using a reversed-phase high per-
formance liquid chromatograph (C18 analytical column with a gradient
of 0–100% B in 45 min; buffer B contained 70% (v/v) aqueous acetoni-
trile, 0.09% trifluoroacetic acid; buffer A contained 0.1% trifluoroacetic
acid). Protein concentrations were determined by Lowry et al. (23), and
purity and homogeneity were assessed by affinity-purified monoclonal
antibody for K5. The antibodies used included mouse anti-K5 (1:1000)
(Chemicon International, Temecula, CA) and sheep monoclonal K5
antibody (1:100) (Binding Site, Birmingham, UK). The availability of a
source of defined and purified K5 has now permitted us to study the
binding properties of this molecule with amelogenins.
Protein Preparations from Ameloblasts—We have isolated amelo-
blasts from mouse postnatal mandibular first molars at different devel-
opmental stages ranging from newborn day “0” through postnatal (PN)
days 1, 3, 5, 7, and 9. Isolated ameloblasts were pooled, frozen, and
thawed (four cycles) to extract the proteins as described with modifica-
tions previously used (20, 21). The cytokeratin fraction was obtained
from cells that were homogenized with approximately three volumes of
ice-cold Tris-HCl (Tris-HCl, pH 7.4, with 1 M urea, 100 ?M EDTA, 10 mM
BME). The homogenates were incubated for about 30 min at 37 °C and
then centrifuged for 5 min at 8,000 ? g in a Beckman Microfuge 12, and
the supernatant was collected. The proteins from the supernatants
were isolated using a Microcon concentrator with a cut-off size of 10,000
and centrifuged at 2,000 ? g for 12 min. The protein fraction was
collected and stored at ?20 °C until further use. Equivalent amounts of
isolated proteins (?20 ?g), as assessed by spectrophotometry (23), were
added to the gel (4–12% gradient SDS-PAGE). After confirming the
homogeneity and purity, the gels were used for Western blot analysis.
Expression and Purification of Recombinant Proteins—Preparation
and purification of recombinant mouse amelogenin rM179 were carried
out as previously described (4, 22).
Preparation and Purification of Synthetic Peptides—All the polypep-
tides (ATMP, T-ATMP, and F-ATMP) used in this study were synthe-
sized by the University of Southern California Microchemical Core
Laboratory using an Applied Biosystems model 430A single-column
peptide synthesizer with the modified Merrifield procedure (24). Pep-
tides were purified as mentioned previously (4, 8).
3H Labeling of ATMP—The 13-residue ATMP “P[3H]YPSYGYE-
PMGGW” was prepared and purified by Amersham Biosciences as
mentioned previously (8).
Western Blot Analysis—K5 purified from HeLa cells and proteins
isolated from ameloblast extracts during different developmental days
(newborn to PN day 9) were resolved by gradient SDS-PAGE (4–12%)
and electrotransferred to polyvinylidene difluoride (PVDF) membranes
(Immobilon-P Transfer Membrane, Millipore Corp.) at 100 mA for 35
min using a semi-dry transblot apparatus (Bio-Rad Scientific Instru-
ments) (5, 8). Protein transfer was assessed by staining the strips with
0.1% Fast Green (Sigma) in 40% methanol and 10% acetic acid (5, 8).
The membranes were washed (five times) and blocked with PBS (pH
7.2) containing ?1% HSA, and were overlaid with appropriate antibod-
ies. The antibodies used included mouse anti-K5 (1:1000, Chemicon
International) and sheep monoclonal K5 antibody (1:100). The Western
blots also were treated with monoclonal antibodies common for both
N-linked and O-linked (IgM antibody, 1:100, Calbiochem) GlcNAc and
with monoclonal antibody specific for O-linked GlcNAc (IgG1, Alexis
Biochemicals, 1:100). Enzyme-linked secondary antibody from Roche
Diagnostics (BM Chemiluminescence Western-blotting kit, 1:2000)
were used. The presence of GlcNAc in proteins was also confirmed by
staining the Western blots with Datura stramonium lectin (8).
Binding of Amelogenin to Varying Concentrations of K5 by Enzyme-
linked Immunosorbent Assay—enzyme-linked immunosorbent assay
was performed using the purified K5 as an antigen following the pro-
tocol described previously (8). Antigen coating was done by adding 100
?l of a solution containing varying amounts of proteins in PBS (pH 7.2)
to microtiter plates (Falcon 3915, Fisher Scientific) and incubating the
plates at 4 °C overnight. Wells were blocked with PBS containing 1%
HSA for 90 min at 37 °C. One-hundred microliters of a known amount
of amelogenin protein (rM179, 5 pmol/100 ?l) was added to wells, and
the mixture was incubated for 1 h at 37 °C. After washing the plates
five times, the primary antibody against the rM179 protein (8) (at a
dilution of 1:6000) (5) was added, and the mixture was incubated for 1 h
at 37 °C and then incubated with a secondary antibody (1:5000, goat
anti-rabbit IgG, Jackson ImmunoResearch, West Grove, PA) for 1 h.
After washing, a substrate (O-phenylenediamine dihydrochloride, In-
vitrogen) in citrate-phosphate buffer and hydrogen peroxidase was
added to the plates for color development. The enzymatic oxidation was
arrested after 30 min in the dark, with 6 N H2SO4. The absorbency
difference at 490–650 nm was measured after automix in a microplate
reader (Molecular Devices, Sunnyvale, CA). The values were corrected
for background (wells without antigen). Ovalbumin was used as a
positive control because it contains two residues of terminal GlcNAc
(25) and BSA as a negative control.
Binding of [3H]ATMP to K5 by Western Blot Analysis and Autora-
diography—Purified K5 was resolved via SDS-PAGE and transferred to
PVDF membranes at 100 mA for 35 min using a semi-dry transblot
apparatus (Bio-Rad Scientific Instruments) (5, 8). Protein transfer was
assessed by staining the PVDF strips with 0.1% Fast Green as men-
tioned previously (5, 8). Replicas were treated with [3H]ATMP (8 ? 105
dpm/ml) and resuspended in PBS (pH 7.2) for 18 h at 25 °C, after
blocking the membrane with PBS containing, 1% HSA for 1 h at 37 °C.
The membranes were washed five times with PBS containing 0.1% HSA
(5, 26). After washing and drying, the membranes were exposed to
hyperfilm-3H (Amersham Biosciences) for 5 days at 4 °C, and the films
were developed manually. To assess whether [3H]ATMP binds GlcNAc
residues of K5, we treated blots of K5 on PVDF membranes, after
blocking with PBS, 1% HSA for 1 h at 37 °C, N-acetyl glucosaminidase
recombinant from Escherichia coli (A6805, Sigma). 100 ?l (5 units) of
the enzyme suspension (in PBS, pH 7.00) was overlaid on two strips.
Control strips were overlaid with blocking buffer. A thin strip of
Parafilm was placed over the solution to retain the enzyme at one
location. The overlaid strips were incubated at room temperature (1 h)
and at 37 °C for 90 min. After washing with the PBS, 0.1% HSA, 0.005%
Tween 20, the strips were incubated in sealer bags with [3H]ATMP (8 ?
105dpm/ml) resuspended in PBS (pH 7.2) for 18 h at 4 °C. The mem-
branes were washed five times with PBS, 0.1% HSA. After washing and
drying, the membranes were exposed to hyperfilm-3H (Amersham Bio-
sciences) for 5 days at 25 °C, and the films were developed manually.
Ovalbumin was used as positive control.
Dosimetric Binding of K5 to [3H]ATMP—100 ?l of [3H]ATMP (30 ?
104dpm in Tris-buffered saline, pH 7.2), was added to 1.5-ml polypro-
pylene microcentrifuge tubes containing increasing amounts of K5 in
TBS (pH 7.2), and the mixture was gently shaken every 20 min for 2 h
at 37 °C as described previously (5, 8). Ovalbumin was used as positive
control and BSA as a negative control.
Specific Binding of K5 to [3H]ATMP as a Function of Increasing
Concentration of ATMP—The total binding of labeled ATMP to K5 (333
by guest on December 22, 2015
J. Biol. Chem.
Basilrose, Sr., Naren H. Ravindranath and
Rajeswari M. H. Ravindranath, Rajam M.
Ameloblasts during Enamel Growth
Amelogenin Interacts with Cytokeratin-5 in
Glycobiology and Extracellular Matrices:
doi: 10.1074/jbc.M211184200 originally published online March 25, 2003
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